PROJECT SUMMARY/ ABSTRACT There is wide recognition within the transplant and broader biomedical communities, as well as government funding agencies, that extended and improved preservation of biological materials is needed for a huge range of endeavors in biomedicine, medical research and drug discovery, organ and tissue transplantation, cell-based therapies, fertility and regenerative medicine, emergency preparedness, and trauma care. The current standard of preservation for organs and vascular composite allografts (VCAs) consists of few hours of hypothermic static storage in UW solution on ice. This contributes to organ/VCA shortage and increased discard rates, exacerbates ischemic injury and graft rejection due to suboptimal donor-recipient matching, and diminishes the quality of life for transplant recipients. To address these challenges, we have pioneered a novel thermodynamic approach to biopreservation, based on a stable equilibrium state and isochoric (constant volume) condition, at high subzero (-5C to -20 C) temperatures, that will allow effective preservation of organs and VCAs, with a 20x-28x increase in storage duration over the current clinical practice (<6-12h of hypothermic storage) while avoiding cellular injury and other challenges created by storage at deep cryogenic temperatures. Importantly, we will build on the successful demonstration of feasibility in Phase I and the results that Sylvatica and UC Berkeley have collaboratively demonstrated with regard to high subzero isochoric preservation: (1) biocompatible thermodynamic pressure profiles for preservation cocktails that support human cell survival in isochoric systems with viabilities above 75%, (2) successful initial scale-up of isochoric preservation to whole rat hearts and skin at unprecedented temperatures (-8C, -10C), and (3) isochoric preservation of fish muscle and an entire organism (the research model C. elegans). Therefore, the objective of this Phase II proposal is to demonstrate, using animal and human models of VCA representative tissues and limb preservation, prolonged cryopreservation of VCAs for 5-7 days and weeks, or longer, with good functional outcome post storage and recovery. Across five specific aims, we will first employ skin and vascular models of VCAs for cryostasis cocktail and protocol optimization, with the central goal of enabling high subzero isochoric preservation while actively suppressing metabolism and enhancing stress tolerance. An isochoric preservation platform (chamber, pressure/temperature sensors) based on the successful system used in Phase I will be designed and built to support cryostasis protocols validation using clinical size human skin and blood vessels, then a model of rat forelimb preservation and assessment through pseudo transplantation, and then by orthotopic allotransplantation with comprehensive characterization of upper extremity functional and behavioral recovery. The results from the animal preservation/transplantation model will be used for the proof-of-concept for high subzero isochoric preservation of human fingers and hands, with full exsanguinous metabolic support quality assessment.